1. Field of the Invention
The present invention relates to a microtissue for inducing the growth of a hair follicle and a method for manufacturing thereof.
2. The Prior Arts
For severe cases of hair loss or alopecia, regeneration of hair follicles (HFs) by bioengineering is a promising future treatment alternative. The combination of tissue engineering techniques with basic biological knowledge, such as stem cell biology and embryonic development, has been advocated recently for reconstruction functional organs or organ germs to restore damaged or lost organs. However, even with various kinds of stem cells available, building new three-dimensional complex microtissues is still very challenging.
Prior arts for HF regeneration can be classified into three methods, including implantation of a large number of dermal papilla cells, induction of HF neogenesis by creating wounds in the skin, and transplantation of folliculoids that are generated in vitro. In the first method, dermal papilla cells (DP cells) must maintain aggregate form during growth to retain function of HFs. Many researches employ this concept with various approaches to promote aggregation of large amount of DP cells then transplanting to the skin of animals. However, the transplantation site of cultured DP cells must be in close proximity to the epidermis to induce HF neogenesis, which increases difficulty in clinical application. In the second method, a suitable size of surgical cut is created on the animal skin to generate an environment that simulating HF development at the embryonic stage. Neogenesis of HFs and hair shafts will appear at the cut site and the regenerated HFs has the characteristics of hair cycle. However, part of the skin must be excised when this method is used and thus clinically unsuitable. Furthermore, this method has not been successfully proved in human. The third method is production of HF microtissues in vitro using polymers or hydrophilic gel as substratum. However, preparation process of such substratum is complicate and successful induction of HF neogenesis is achieved when embryonic or newborn cells are employed.
As described in Taiwan Patent Publication Nos. 200800240 and 200539842, co-existence of keratinocytes and DP cells can enhance neogenesis of HFs, however, these are known facts for more than ten years and practical applications of these techniques are still challenging and encountering many difficulties.
Researches have shown that DP cells on the EVAL (poly (ethylene-co-vinyl alcohol) membrane can aggregate to form three dimensional microtissues and extracellular matrix protein coated on the EVAL membrane may induce formation of DP cell microtissues. Moreover, previous publication (Quiao Z et al. Regenerative Medicine 2008) has shown that embryonic mouse skin cells under hanging drop cultivation condition are able to develop into hair-like structures in vitro. When transplanted into the mouse, these hair-like structures can further grow into mature HFs. However, this method is not successful when adult cells are used. Another publication (Havlickova et al. J Invest Dermatol. 2009) points out that, when DP cells and keratinocytes are mixed with extracellular matrix, these cells can develop into jelly-like microtissues. However, this method fails to develop microtissues into mature HFs.
It is well-known that adult keratinocytes do not respond well to inductive signals from cultured DP cells to generate new HFs unless keratinocyte stem cells are used. However, keratinocyte stem cells can be collected only by excision and separation of existing viable HFs. Furthermore, this approach not only harvests scarce amount of cells but also sacrifices more HFs. Therefore, it is not possible to use this method for limited amount of new HFs in clinical applications.